Stimulation of glucose transport in skeletal muscle by hypoxia GREGORY D. CARTEE, ANDRE G. DOUEN, TOOLSIE RAMLAL, AMIRA KLIP, AND JOHN 0. HOLLOSZY Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110; Biodynamics Laboratory, University of Wisconsin, Madison, Wisconsin 53706; and Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5GlX8, Canada CARTEE,GREGORY D., ANDREG.DOUEN,TOOLSIERAMLAL, tion, such as the increase in cytosolic Ca2+concentration AMIRA KLIP, ANDJOHN 0. HOLLOSZY.Stimulation ofglucose or the utilization of high-energy phosphates, without also transport in skeletal muscle by hypoxia. J. Appl. Physiol. 70(4): affecting the contractile process itself. We have, there1593-1600, 1991.-Hypoxia caused a progressive cytochalasin fore, become interested in the possibility that hypoxia, B-inhibitable increase in the rate of 3-0-methylglucose transwhich has been shown to increase cytosolic Ca2+concenport in rat epitrochlearis muscles to a level approximately sixtration in heart muscle (38), may stimulate glucose transfold above basal. Muscle ATP concentration was well mainport in muscle via the same pathway as contractile activtained during hypoxia, and increased glucose transport activity ity, as this could provide a simpler model in which to was still present after 15 min of reoxygenation despite replestudy the mechanisms responsible for the increase in tion of phosphocreatine. However, the increase in glucose transglucose transport activity. port activity completely reversed during a NO-min-long recovery in oxygenated medium. In perfused rat hindlimb muscles, In this context, we have studied the effects of hypoxia hypoxia caused an increase in glucose transporters in the on 3-MG transport in rat skeletal muscle. We found that plasma membrane, suggesting that glucose transporter transhypoxia causes a large stimulation of 3-MG transport in location plays a role in the stimulation of glucose transport by epitrochlearis muscles and that the maximal effects of hypoxia. The maximal effects of hypoxia and insulin on glucose hypoxia and insulin on 3-MG transport are roughly additransport activity were additive, whereas the effects of exercise tive. In contrast, the maximal effect of hypoxia and conand hypoxia were not, providing evidence suggesting that hyptractile activity together are no greater than that of eioxia and exercise stimulate glucose transport by the same mechther stimulus alone, suggesting that contractile activity anism. Caffeine, at a concentration too low to cause muscle and hypoxia increase glucose transport by the same contraction or an increase in glucose transport by itself, markmechanism. Caffeine at a concentration that had no efedly potentiated the effect of a submaximal hypoxic stimulus fect by itself potentiated the effect of a submaximal hypon sugar transport. Dantrolene significantly inhibited the hypoxia-induced increase in 3-0-methylglucose transport. These oxic stimulus, whereas dantrolene, an inhibitor of Ca2+ effects of caffeine and dantrolene suggest that Ca2’ plays a role release from the sarcoplasmic reticulum (7), partially inin the stimulation of glucose transport by hypoxia. hibited the effect of hypoxia on sugar transport. These findings support the hypothesis that Ca2+plays a role in calcium; epitrochlearis muscle; exercise; insulin; 3-O-methylthe activation of glucose transport by a number of stimglucose uli, including hypoxia and muscle contraction (2,13,14). IN MUSCLE CELLS, glucose transport is stimulated by insulin and by agents that increase the demand for glucose. The latter include muscle contractions (1 l), hypoxia (30, 33), and poisons that uncouple or inhibit respiration (22, 33). Available information suggests that muscle contractions and insulin stimulate glucose transport by separate pathways. Perhaps the strongest evidence for this is that the maximal effects of insulin and contractile activity on transport of the nonmetabolizable glucose analogues 3OLmethylglucose (3-MG) or 2deoxyglucose are roughly additive in mammalian skeletal muscle (3, 29, 42). Because of the important role that exercise plays in muscle glucose metabolism, the mechanism by which muscle contractions stimulate glucose transport is of considerable interest. However, studies of this process have been seriously limited by the inability to experimentally manipulate the events associated with muscle contrac-
METHODS Treatment of animals. Male specific-pathogen-free Sprague-Dawley rats were obtained from SASCO (Omaha, NE) and maintained on a diet of Purina chow and water ad libitum. In some experiments, rats were exercised by swimming intermittently for 2 h as described previously (42). Muscle preparation and incubation. After an overnight fast, rats weighing -160 g were anesthetized with 5 mg/ 100 g body wt of pentobarbital sodium, and the epitrochlearis muscles were dissected out. In contrast to adipose tissue, fasting does not cause insulin resistance of the glucose transport process in rat skeletal muscle (Ref. 39 and unpublished observations). Muscle weights averaged -20 mg. The epitrochlearis is a thin muscle only -22 fibers thick, and this short diffusion distance makes it suitable for in vitro studies (28,46). It is made up predom1593
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inantly of fasttwitch fibers (28). The muscles were incubated in a shaking incubator at 35°C in 2 ml Krebs-Henseleit bicarbonate buffer (KHB) (24) that contained 0.1% bovine serum albumin, 32 mM mannitol, 8 mM glucose, and various additions in stoppered Erlenmeyer flasks with a gas phase of either 95% O,-5% CO, or 95% N,-5% CO, (hypoxia). To keep osmolarity constant throughout an experiment, sufficient mannitol was included in the medium to keep the sum of the concentrations of glucose or 3-MG plus mannitol at 40 mM. Specific information regarding the additions to the incubation medium is provided for each experiment. Stimulation of muscle contraction. The muscle’s distal tendon was attached to a vertical Lucite rod containing two platinum electrodes (12). The proximal tendon was clipped to a jeweller’s chain that connected it to a force transducer (Grass model FTOC), and resting tension was adjusted to 0.5 g. The mounted muscle was immersed in 20 ml of KHB maintained at 35°C and continuously oxygenated with 95% O,-5% CO,. The muscles were stimulated wit.h supramaximal square-wave pulses of 0.2-ms duration with a Grass S48 stimulator. The tetanic contractions were elicited by stimulating at 100 Hz for 10 s at a rate of 1 contraction/min for 10 min. When muscles were stimulated after incubation in hypoxic medium, they were allowed to recover in oxygenated medium for 5 min before stimulation of contraction. Measurement of glucose transport activity. Unless otherwise stated, after the experimental intervention (i.e., incubation in hypoxic medium, contractions, treatment with insulin) muscles were placed in oxygenated KHB containing 2 mM Na pyruvate and incubated at 29OCin a shaking incubator for 15 min to wash out glucose and reoxygenate hypoxic muscles. 3-MG transport was measured by a modification (46) of the procedure used previously in frog muscle (12). The muscles were blotted and transferred to a flask with 1.5 ml of KHB containing 8 mM 3-O-[methyl-3H]glucose (437 &i/mmol) and 32 mM [14C]mannitol (8 &i/mmol) and incubated at 29°C in a shaking incubator. Unless otherwise stated, the gas phase was 95% O,-5% CO,. After a brief period in which the extracellular space equilibrates with the medium, 3MG uptake is linear until the intracellular concentration of the extracellular concentration (46). reaches -25% After incubation the muscles were processed, and intracellular 3-MG concentration was determined and expressed as micromoles of 3-MG per milliliter of intracellular water, as previously described (46). Hindlimb muscleperfusion. To obtain sufficient muscle for studies of glucose transporter translocation (-30 g of muscle was required per preparation), rats weighing -375 g were fasted overnight and then anesthetized with 5 mg/lOO g body wt of pentobarbital sodium and prepared for hindlimb perfusion with placement of catheters in the abdominal aorta and vena cava (35). The rats’ hindlimb musculature and the epitrochlearis muscle are composed predominately (85-95%) of fast-twitch fibers (1, 28). The perfusion apparatus and procedure have been described previously (34). The perfusion medium consisted of KHB containing 4 g/100 ml of bovine serum albumin and 8 mM glucose. The perfusate flow rate was 20 ml/min. The perfusion medium and hindquarter prep-
IN
MUSCLE
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aration were kept at 37OC. The effect of hypoxia on glucose uptake was determined during 40-min-long flowthrough perfusions with medium gassedwith 95% N,-5% CO,. Arterial and venous samples of perfusate were taken after 10 min (oxygenated controls) or 40 min (hypoxic) of perfusion. The control value was obtained after 10 min because of concern that the muscles might not be adequately oxygenated in the absence of erythrocytes in the perfusate for 40 min; the glucose uptake rate after 10 min of perfusion with oxygenated medium in the absence of erythrocytes was essentially the same as that obtained in hindlimbs perfused for 30-60 min with oxygenated medium containing erythrocytes. The arteriovenous glucose concentration differences and perfusate flow rate were used to calculate the rate of glucose uptake. Preparation of membrane fraction. After the perfusion the hindlimb muscles were dissected out, placed on ice, and rapidly cleaned of fat and connective tissue and then clamp-frozen with aluminum tongs cooled in liquid N,. Muscles were kept at -70°C until used for preparation of membranes. The procedure for the isolation of plasma membrane and internal membrane fractions has been described in detail (20). Briefly, -30 g of rat hindlimb muscles were minced in 250 mM sucrose containing 10 mM NaHCO,, pH 7.0, and then subjected to Polytron homogenization. The homogenate was subjected to a series of centrifugation steps to yield a crude pellet (20). Membrane fractions were then separated by 16 h of centrifugation on discontinuous sucrose gradients (25% and 35% sucrose, wt/wt). Membranes were collected from both sucrose layers, washed by IO-fold dilution in the homogenizing medium, and recovered by high-speed centrifugation. Samples were immediately assayed for cytochalasin B binding (20). The remainder was frozen and stored at -2OOC and used within 1 wk for measurement of Y-nucleotidase activity (19) or for subsequent Western-blot analysis as described previously (6). Muscle glucose transporter protein of the GLUT-4 type’ was detected using the antiserum R820 (16) and 1251-labeled protein A as described previously (6). The R820 antiserum was a kind gift from Dr. David E. James, Washington University, St. Louis, MO. The autoradiographs were quantified by laser-scanning densitometry. Assay methods. Epitrochlearis muscles to be used for measurement of ATP and creatine phosphate content were clamp-frozen with tongs cooled in liquid N,. Weighed portions of frozen muscle were homogenized in HClO, (27). An aliquot of this homogenate was used for determination of glycogen by the amyloglucosidase method (32). The remaining homogenate was centrifuged at 5,000 g for 10 min at 4°C. The supernatant was neutralized and used for measurement of ATP (26) and phosphocreatine (25). Statistics. The statistical significance of differences between groups was determined using either Student’s t test or, when more than two groups were compared, by ’ The nomenclature of Graeme I. Bell’s group (Diabetes Cure 13: 1984208, 1990) is used to identify glucose transporter subtypes. In this nomenclature, GLUT-l is the glucose transporter cloned from HepG2/ rat brain and GLUT-4 is the transporter from rat and human muscle and adipocytes.
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STIMULATION
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1595
transport activity was still present after a recovery period sufficiently long to permit repletion of the muscles’ phosphocreatine store (see below). However, the in% 1.2 7 crease in glucose transport activity completely reversed d -7 during a 180-min-long recovery in oxygenated medium; E-E lo. the 3-MG transport rate averaged 1.19 t 0.07 WO pm01 ml-l 10 min-’ in 12 muscles studied after 15 min m-r 08. 0 of recovery from hypoxia and 0.20 t 0.02 pmol . ml-’ 10 y: ()6min-’ in 12 muscles allowed to recover from hypoxia for 6% E * 180 min in oxygenated medium containing 8 mM glucose. -r1 04f The return to baseline of sugar transport activity during >I a 180-min-long recovery period in oxygenated medium 0.2 - #----~----r---------f ---Iw was not altered by addition of 20 ,ug/ml of cycloheximide z 1 I 1 I 1 1 to the incubation medium (0.19 t 0.08 prnol. ml-’ A 80 100 0 20 40 60 10 min-‘). TIME (min) Effects of hypoxia on muscleATP, phosphocreatine, and glycogen concentrations. Phosphocreatine concentration FIG. 1. Time course of increase in 3-O-methylglucose transport rate in response to hypoxia. Rat epitrochlearis muscles were incubated decreased to -34% of the oxygenated control value in under either oxygenated or hypoxic conditions for various time interepitrochlearis muscles during 80 min of hypoxia (Table vals at 35°C and then for an additional 15 min in .oxygenated medium 2); most of this decrease occurred during the first 20 min at 29°C. 30methylglucose transport rate was then measured at 29°C of hypoxia. There was no appreciable decline in ATP as described (see METHODS). Values are means + SE for 4-8 muscles and are expressed as pmol of 3-0-methylglucose taken up per ml of concentration during 80 min of hypoxia. Muscle glycogen intracellular water in 10 min. concentration declined -66% during 80 min of hypoxia. Phosphocreatine concentration returned to the oxygenanalysis of variance. A Newman-Keuls post hoc test was ated control level within 15 min in muscles allowed to used to locate the source of significant variance. recover from hypoxia in oxygenated medium. A4ateriaLs. ICN Radiochemicals was the source for Hypoxia-induced translocation of glucosetransporters in 3-O- [ methyl-3H] glucose and [ 14C]mannitol. [3H]cytorat hindlimb muscles.To obtain sufficient muscle to prechalasin B was from Amersham. Sodium dantrolene was pare membrane fractions for the study of cytochalasin B obtained from Norwich Eaton Pharmaceuticals. Purified binding, we used the perfused rat hindquarter preparapork insulin was purchased from Squibb. Bovine serum tion. Perfusion of the hindlimb muscles for 40 min with albumin (Cohn Fraction V, fatty acid poor) was obtained hypoxic medium caused an increase in the rate of glucose from Miles Laboratories. All other reagents were pur- uptake to 20.5 t 1.1 pmol/min per hindquarter compared chased from Sigma. with 3.4 t 1.2 pmollmin per hindquarter for the controls (P < 0.01). (For comparison, the rate of glucose uptake in RESULTS rat hindquarters after 30-60 min of perfusion with oxy3-0-methylglucose transport. Hypoxia caused a large genated medium containing human erythrocytes at a concentration of hemoglobin of 12 g/100 ml in two stimulation of glucose transport activity in epitrochlearis muscles. As shown in Fig. 1, the increase in the rate of previous studies (15,45) averaged 3.3 t 0.3 pmol/min per 3-MG transport in response to hypoxia occurred over hindquarter for 33 rats.) The crude membrane fraction obtained from -30 g of -60 min, plateauing at a level approximately sixfold hindlimb muscle was subjected to discontinuous sucroseabove baseline. This hypoxia-induced increase in 3-MG uptake was due to activation of the glucose transport TABLE 1. Effects of cytochalasin B and of cycloheximide process and not to a nonspecific increase in permeability. on stimulation of 3-0-methylglucose transport by hypoxia This is evidenced by the findings that 1) 25 PM cytochalasin B completely inhibited 3-MG transport after 80 min in rat epitrochlearis muscle of hypoxia (Table 1) and 2) the muscles remained imper3-O-Methylglucose Transport, meable to mannitol. Cycloheximide (20 pg/ml), which inpm01 . ml ’ . 10 min.-’ Treatment I1 hibits protein synthesis in epitrochlearis muscle by 20 0.16+0.02 Control oxygenated -95% (46), had no effect on the increase in permeability 25 1.18t0.06 Hypoxia to 3-MG induced by hypoxia (Table 1). 0.0:3-to.o2 Hypoxia + 25 PM cytochalasin B 5 In the above experiments the muscles were allowed to Hypoxia -t 20 pg/ml cycloheximide 1.25kO.16 5 recover for 15 min in oxygenated medium, and 3-MG Values are means + SE and are expressed as pmol of 3-MG taken up transport was measured under aerobic conditions. When per ml of intracellular water in 10 min; IZ, no. of muscles. Epitrochlearis muscles were kept hypoxic throughout an 80-min-long muscles were incubated under oxygenated or hypoxic conditions with 8 incubation and the subsequent glucose washout and 3- mM glucose for 80 min at 35°C and were transferred to oxygenated MG transport measurement periods, the rate of 3-MG medium containing no glucose and incubated at 29°C for 15 min to wash out glucose and reoxygenate hypoxic muscles. Glucose transport transport was not significantly higher than in paired accumulation of muscles allowed to recover for 15 min in oxygenated me- activity was then assessed by measuring intracellular 3-0-methylglucose (3-MG) for 10 min at 29°C in oxygenated medium. dium before measurement of 3-MG transport (1.39 t In 1 experiment, cytochalasin B was present during wash period and 0.08 vs. 1.33 t 0.14 pmol . ml-’ 10 min-’ for 9 muscles measurement of 3-MG transport. In another experiment, cyclohexiincubations. per group). Thus the hypoxia-induced increase in glucose mide was present throughout
L 0
14.
l
l
l
l
l
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2. Effects of hypoxia on epitrochlearis muscle ATP, phosphocreatine, and glycogen concentrations Treatment
pmol/g
Oxygenated control Hypoxia, 20 min Hypoxia, 80 min Reoxygenated
ATP, protein
Phosphocreatine, pmol/g protein
29.8t0.8 28.1k1.3 29.6k2.9
(18) (7) (5)
93.8+6.0 41.8t2.9* 32.2t3.7*
(18) (7) (5)
28.121.1
(11)
91.5k7.3
(11)
Glycogen, pmol/g protein
179+4 143+8 61-t12*
(8) (7) (5)
Values are means + SE for no. of muscles given in parentheses. Epitrochlearis muscles were incubated for 20 or 80 min under oxygenated or hypoxic conditions with 8 mM glucose at 35°C and then clamp-frozen. Additional muscles that had been incubated for 80 min under hypoxic conditions were transferred to oxygenated medium (reoxygenated) and incubated for 15 min before being frozen. Because there was no difference between values obtained on muscles incubated in oxygenated medium for 80 compared with 20 min, data obtained on these 2 groups were combined (oxygenated control). *Significantly less than oxygenated control (P < 0.01).
gradient centrifugation. The membrane fraction recovered floating on 25% sucrose is enriched approximately fivefold in the activities of the plasma membrane marker enzymes 5’-nucleotidase (Table 3 and Ref. ZO), Mg2+-ATPase (20)) and phosphodiesterase (20) relative to the crude membrane fraction, whereas the 35% sucrose fraction is relatively poor in these marker enzyme activities (Table 3 and Ref. 20). Sarcoplasmic reticulum Ca2+-ATPase activity was not detectable in either fraction. We have previously found that the number of glucose transporters, as measured by D-glucose-inhibitable cytochalasin B binding (20) and immunoblotting for the GLUT-4 glucose transporter (6), is increased in the 25% sucrose fraction and decreased in the 35% sucrose fraction from muscles treated with insulin. Taken together these findings provide evidence that the 25% sucrose fraction is enriched with plasma membrane, whereas the 35% sucrose fraction is poor in plasma membranes and probably contains an intracellular membranous organelle that, as in adipocytes (4,40), contains glucose transporters. Figure 2 shows the D-glucose-inhibitable cytochalasin B binding data expressed per milligram of protein; the relationships are the same when the data are expressed as the total amount of D-glucose-inhibitable cytochalasin B binding recovered in each fraction. Hypoxia resulted in an -50% increase in D-glucose-inhibitable cytochalasin B binding in the 25% sucrose (plasma membraneenriched) fraction, suggesting that hypoxia causes a translocation of glucose transporters into the plasma membrane in skeletal muscle. The difference in D-gh-
case-inhibitable cytochalasin B binding in the 35% sucrose fractions from the hypoxic and control muscles was not statistically significant. The effect of hypoxia on distribution of the GLUT-4 muscle glucose transporters in the plasma membrane and intracellular membrane fractions was also examined. Plasma membrane and intracellular membrane fractions prepared from control and hypoxic muscles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (30 fig protein from each fraction), transferred to nitrocellulose, and immunoblotted with anti-GLUT-4 antibody (Fig. 3). Hypoxia increased the amount of GLUT-4 transporters in the plasma membrane fraction but had little effect on the GLUT-4 content of the intracellular membrane fraction (Fig. 3). In two independent experiments, each analyzed twice, quantification by laser-scanning densitometry showed that hypoxia caused an average 2.4fold increase in GLUT-4 transporters in the 25% sucrose plasma membrane fraction without a decrease in GLUT-4 transporters in the 35% sucrose intracellular membrane fraction (Fig. 4). Interactions between hypoxia, muscle contractile activity, and insulin. As shown in Table 4, the maximal effects of in vitro muscle contractions or swimming on 3-MG transport were not significantly different from that of hypoxia. Furthermore, the combined effect of a maximal
20. 3. Protein content and 5’-nucleotidase activity in membrane fractions isolated from rat hindquarter muscles
TABLE
0 E a
5’-Nucleotidase, nmol min-’ protein-’ l
Protein,
mg/fraction
l
mg
Fraction
Control
Hypoxia
Control
Hypoxia
Crude membranes 25% Sucrose 35% Sucrose
42.2k6.2 1.26kO.13 ll.Ok1.8
38.5k5.2 1.2MO.08 10.3+_0.9
85+4 424k14 73+8
66+6 348+40 52+_5
Values are means k SE for 6 independent membrane preparations. Crude membrane fraction obtained from -30 g of rat hindlimb muscles was subjected to discontinuous sucrose gradient centrifugation (see METHODS). Aliquots of crude membrane preparation and membrane fractions recovered floating on 25% sucrose and on 35% sucrose were used for measurement of 5’-nucleotidase activity.
F ‘c1 1.0 c c1 m 0 00. 25% Membrane
3 5% Fraction
FIG. 2. Cytochalasin B (CB, 0.2 pM) binding to the 25% sucrose (plasma membrane) and 35% sucrose (internal membranes) fraction isolated from control and hypoxic muscles. Values are means + SE for 6 independent membrane preparations.
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STIMULATION
PM -II-II ’
OF GLUCOSE
IM
TRANSPORT
PM
IN MUSCLE
IM
1-- ,,’ :
43 kDa__>
r
._. \
“,
1597
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\ __ _C_\\\_
CHCHCH
C
hypoxic stimulus and in vitro contractile activity on 3MG transport was not significantly different from that of either stimulus alone. Similarly, muscles taken from rats after a bout of swimming and then incubated under hypoxic conditions in vitro showed no greater increase in 3-MG transport than that induced by swimming alone (Table 4). This lack of additivity suggests that hypoxia and muscle contractions may activate glucose transport by the same mechanism. It has been shown previously (3, 29, 42) and again in this study (Table 5) that the maximal effects of exercise and insulin on glucose transport activity (i.e., increases above basal) are roughly additive in rat skeletal muscle. As would be expected if hypoxia and muscle contractions activate glucose transport by the same mechanism, the increases above basal in glucose transport activity induced by 60 min of hypoxia and a maximally effective insulin concentration were also additive (Table 5). Potentiation of a submaximal hypoxic stimulus by caffeine and nitrate. It has been hypothesized on the basis of
indirect evidence that the increase in cytoplasmic Ca2+ that occurs in response to stimulation of muscle contraction is the initiating event in the process that leads to activation of glucose transport by contractile activity
H
FIG. 3. Effect of hypoxia on subcellular distribution of the GLUT-4 transporter in skeletal muscle membranes. Plasma membranes (PM) and intracellular membranes (IM) were prepared from control (C) and hypoxic (H) rat hindlimb muscles. Membranes (30 pg protein) were separated on 12% polyacrylamide gels, transferred to nitrocellulose paper, and immunoassaved bv Western blot analvsis usine anti-GLUT-4 a&erum (see METHODS). Shown is an experiment with 2 independent membrane preparations.
(13). As a preliminary approach to evaluating the possibility that an increase in Ca2+ also mediates the activation of glucose transport by hypoxia, we examined the effects of caffeine and NO,. Caffeine induces release of Ca2+ from the sarcoplasmic reticulum (31) and, at sufficiently high concentration, causes muscle contraction and stimulation of glucose transport activity (13). In the rat epitrochlearis, a caffeine concentration above -3 mM is required to induce measurable tension development. Incubation of epitrochlearis muscles with 10 mM caffeine for 30 min induced an increase in 3-MG transport rate to 1.03 * 0.12 compared with a control value of 0.17 * 0.03 pm01 * ml-’ -10 min-’ (means + SE for 9 muscles per group); this is similar in magnitude to the response to a maximal hypoxic stimulus. Low concentrations of caffeine can elicit increases in cytosolic free-Ca2+ concentration that are below the contraction threshold (23). Incubation with 1 mM caffeine had no effect on 3-MG transport in oxygenated muscles but caused a significant potentiation of the effect of a submaximal (40-min-long) hypoxic stimulus on the rate of 3-MG transport without causing a contracture (Table 6) to give a sugar transport rate similar to that seen after a maximal hypoxic stimulus. However, 1 mM caffeine did not potentiate the effect of a maximal (80-min-long) hypoxic stimulus. NO, impairs the active uptake of Ca2+ by the sarcoplasmic reticulum and, as a consequence, potentiates muscle twitch tension (7). NO, also potentiates a submaximal effect of muscle contractions on glucose transport activity (14). Incubation of muscle in KHB in which 4. Effects of hypoxia and muscle contractile activity together on 3-O-methylglucose transport TABLE
Treatment
PM
IM membrane
fraction
FIG. 4. Effect of hypoxia on subcellular distribution of GLUT-4 transporters in skeletal muscle PM and IM preparations. OD, optical density. Values are expressed as a relative increase in response to hypoxia. Two independent membrane preparations were analyzed. Each preparation was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoassay by Western blot analysis twice. Each blot was developed twice, and each X-ray film was densitometritally scanned twice. Values are averages f SD of these determinations.
Oxygenated control Hypoxia In vitro muscle contractions Hypoxia + muscle contractions Swimming Swimming + hypoxia
It
11 11 12 9 17 15
3-0-Methylglucose jmol~ ml-‘.
Transport, 10 min-’
0.21*0.02 1.18+0.10 1.15rto.07 1.34kO.08 1.40+0.14 1.31+0.13
Values are means + SE; n, no. of muscles. Epitrochlearis muscles were incubated, under oxygenated or hypoxic conditions, with 8 mM glucose for 60 min at 35°C. In 1 series of experiments, muscles were transferred to oxygenated medium for 5 min and then stimulated to contract (see METHODS). In a second series of experiments, muscles were from rats that had been exercised by means of swimming immediately before study.
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Oxygenated control Hypoxia 2,000 PUlml insulin Hypoxia + 2,000 pU/ml insulin Swimming Swimming + 2,000 &J/ml insulin
n
12 12 8 8 6 6
O.ZOrtO.03 1.2O-tO.08 1.26kO.09 2.22+0.13* 1.22+0.15 1.98*0.09j-
NaNO, was substituted for NaCl had no effect on glucose transport activity when the medium was oxygenated (Table 6). However, NO, significantly potentiated the effect of a submaximal (40 min) hypoxic stimulus on glucose transport (Table 6). The maximal response to hypoxia was unaffected by NO,. Effect of dantrolene. Dantrolene inhibits release of Ca2+ from the sarcoplasmic reticulum in skeletal muscle (7). As shown in Table 7, 25 PM dantrolene partially inhibited the hypoxia-induced stimulation of glucose transport activity. The increase in 3-MG transport rate in response to 60 min of hypoxia was
METHODS Treatment of animals. Male specific-pathogen-free Sprague-Dawley rats were obtained from SASCO (Omaha, NE) and maintained on a diet of Purina chow and water ad libitum. In some experiments, rats were exercised by swimming intermittently for 2 h as described previously (42). Muscle preparation and incubation. After an overnight fast, rats weighing -160 g were anesthetized with 5 mg/ 100 g body wt of pentobarbital sodium, and the epitrochlearis muscles were dissected out. In contrast to adipose tissue, fasting does not cause insulin resistance of the glucose transport process in rat skeletal muscle (Ref. 39 and unpublished observations). Muscle weights averaged -20 mg. The epitrochlearis is a thin muscle only -22 fibers thick, and this short diffusion distance makes it suitable for in vitro studies (28,46). It is made up predom1593
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1594
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TRANSPORT
inantly of fasttwitch fibers (28). The muscles were incubated in a shaking incubator at 35°C in 2 ml Krebs-Henseleit bicarbonate buffer (KHB) (24) that contained 0.1% bovine serum albumin, 32 mM mannitol, 8 mM glucose, and various additions in stoppered Erlenmeyer flasks with a gas phase of either 95% O,-5% CO, or 95% N,-5% CO, (hypoxia). To keep osmolarity constant throughout an experiment, sufficient mannitol was included in the medium to keep the sum of the concentrations of glucose or 3-MG plus mannitol at 40 mM. Specific information regarding the additions to the incubation medium is provided for each experiment. Stimulation of muscle contraction. The muscle’s distal tendon was attached to a vertical Lucite rod containing two platinum electrodes (12). The proximal tendon was clipped to a jeweller’s chain that connected it to a force transducer (Grass model FTOC), and resting tension was adjusted to 0.5 g. The mounted muscle was immersed in 20 ml of KHB maintained at 35°C and continuously oxygenated with 95% O,-5% CO,. The muscles were stimulated wit.h supramaximal square-wave pulses of 0.2-ms duration with a Grass S48 stimulator. The tetanic contractions were elicited by stimulating at 100 Hz for 10 s at a rate of 1 contraction/min for 10 min. When muscles were stimulated after incubation in hypoxic medium, they were allowed to recover in oxygenated medium for 5 min before stimulation of contraction. Measurement of glucose transport activity. Unless otherwise stated, after the experimental intervention (i.e., incubation in hypoxic medium, contractions, treatment with insulin) muscles were placed in oxygenated KHB containing 2 mM Na pyruvate and incubated at 29OCin a shaking incubator for 15 min to wash out glucose and reoxygenate hypoxic muscles. 3-MG transport was measured by a modification (46) of the procedure used previously in frog muscle (12). The muscles were blotted and transferred to a flask with 1.5 ml of KHB containing 8 mM 3-O-[methyl-3H]glucose (437 &i/mmol) and 32 mM [14C]mannitol (8 &i/mmol) and incubated at 29°C in a shaking incubator. Unless otherwise stated, the gas phase was 95% O,-5% CO,. After a brief period in which the extracellular space equilibrates with the medium, 3MG uptake is linear until the intracellular concentration of the extracellular concentration (46). reaches -25% After incubation the muscles were processed, and intracellular 3-MG concentration was determined and expressed as micromoles of 3-MG per milliliter of intracellular water, as previously described (46). Hindlimb muscleperfusion. To obtain sufficient muscle for studies of glucose transporter translocation (-30 g of muscle was required per preparation), rats weighing -375 g were fasted overnight and then anesthetized with 5 mg/lOO g body wt of pentobarbital sodium and prepared for hindlimb perfusion with placement of catheters in the abdominal aorta and vena cava (35). The rats’ hindlimb musculature and the epitrochlearis muscle are composed predominately (85-95%) of fast-twitch fibers (1, 28). The perfusion apparatus and procedure have been described previously (34). The perfusion medium consisted of KHB containing 4 g/100 ml of bovine serum albumin and 8 mM glucose. The perfusate flow rate was 20 ml/min. The perfusion medium and hindquarter prep-
IN
MUSCLE
BY
HYPOXIA
aration were kept at 37OC. The effect of hypoxia on glucose uptake was determined during 40-min-long flowthrough perfusions with medium gassedwith 95% N,-5% CO,. Arterial and venous samples of perfusate were taken after 10 min (oxygenated controls) or 40 min (hypoxic) of perfusion. The control value was obtained after 10 min because of concern that the muscles might not be adequately oxygenated in the absence of erythrocytes in the perfusate for 40 min; the glucose uptake rate after 10 min of perfusion with oxygenated medium in the absence of erythrocytes was essentially the same as that obtained in hindlimbs perfused for 30-60 min with oxygenated medium containing erythrocytes. The arteriovenous glucose concentration differences and perfusate flow rate were used to calculate the rate of glucose uptake. Preparation of membrane fraction. After the perfusion the hindlimb muscles were dissected out, placed on ice, and rapidly cleaned of fat and connective tissue and then clamp-frozen with aluminum tongs cooled in liquid N,. Muscles were kept at -70°C until used for preparation of membranes. The procedure for the isolation of plasma membrane and internal membrane fractions has been described in detail (20). Briefly, -30 g of rat hindlimb muscles were minced in 250 mM sucrose containing 10 mM NaHCO,, pH 7.0, and then subjected to Polytron homogenization. The homogenate was subjected to a series of centrifugation steps to yield a crude pellet (20). Membrane fractions were then separated by 16 h of centrifugation on discontinuous sucrose gradients (25% and 35% sucrose, wt/wt). Membranes were collected from both sucrose layers, washed by IO-fold dilution in the homogenizing medium, and recovered by high-speed centrifugation. Samples were immediately assayed for cytochalasin B binding (20). The remainder was frozen and stored at -2OOC and used within 1 wk for measurement of Y-nucleotidase activity (19) or for subsequent Western-blot analysis as described previously (6). Muscle glucose transporter protein of the GLUT-4 type’ was detected using the antiserum R820 (16) and 1251-labeled protein A as described previously (6). The R820 antiserum was a kind gift from Dr. David E. James, Washington University, St. Louis, MO. The autoradiographs were quantified by laser-scanning densitometry. Assay methods. Epitrochlearis muscles to be used for measurement of ATP and creatine phosphate content were clamp-frozen with tongs cooled in liquid N,. Weighed portions of frozen muscle were homogenized in HClO, (27). An aliquot of this homogenate was used for determination of glycogen by the amyloglucosidase method (32). The remaining homogenate was centrifuged at 5,000 g for 10 min at 4°C. The supernatant was neutralized and used for measurement of ATP (26) and phosphocreatine (25). Statistics. The statistical significance of differences between groups was determined using either Student’s t test or, when more than two groups were compared, by ’ The nomenclature of Graeme I. Bell’s group (Diabetes Cure 13: 1984208, 1990) is used to identify glucose transporter subtypes. In this nomenclature, GLUT-l is the glucose transporter cloned from HepG2/ rat brain and GLUT-4 is the transporter from rat and human muscle and adipocytes.
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STIMULATION
OF
GLUCOSE
TRANSPORT
IN
MUSCLE
BY
HYPOXIA
1595
transport activity was still present after a recovery period sufficiently long to permit repletion of the muscles’ phosphocreatine store (see below). However, the in% 1.2 7 crease in glucose transport activity completely reversed d -7 during a 180-min-long recovery in oxygenated medium; E-E lo. the 3-MG transport rate averaged 1.19 t 0.07 WO pm01 ml-l 10 min-’ in 12 muscles studied after 15 min m-r 08. 0 of recovery from hypoxia and 0.20 t 0.02 pmol . ml-’ 10 y: ()6min-’ in 12 muscles allowed to recover from hypoxia for 6% E * 180 min in oxygenated medium containing 8 mM glucose. -r1 04f The return to baseline of sugar transport activity during >I a 180-min-long recovery period in oxygenated medium 0.2 - #----~----r---------f ---Iw was not altered by addition of 20 ,ug/ml of cycloheximide z 1 I 1 I 1 1 to the incubation medium (0.19 t 0.08 prnol. ml-’ A 80 100 0 20 40 60 10 min-‘). TIME (min) Effects of hypoxia on muscleATP, phosphocreatine, and glycogen concentrations. Phosphocreatine concentration FIG. 1. Time course of increase in 3-O-methylglucose transport rate in response to hypoxia. Rat epitrochlearis muscles were incubated decreased to -34% of the oxygenated control value in under either oxygenated or hypoxic conditions for various time interepitrochlearis muscles during 80 min of hypoxia (Table vals at 35°C and then for an additional 15 min in .oxygenated medium 2); most of this decrease occurred during the first 20 min at 29°C. 30methylglucose transport rate was then measured at 29°C of hypoxia. There was no appreciable decline in ATP as described (see METHODS). Values are means + SE for 4-8 muscles and are expressed as pmol of 3-0-methylglucose taken up per ml of concentration during 80 min of hypoxia. Muscle glycogen intracellular water in 10 min. concentration declined -66% during 80 min of hypoxia. Phosphocreatine concentration returned to the oxygenanalysis of variance. A Newman-Keuls post hoc test was ated control level within 15 min in muscles allowed to used to locate the source of significant variance. recover from hypoxia in oxygenated medium. A4ateriaLs. ICN Radiochemicals was the source for Hypoxia-induced translocation of glucosetransporters in 3-O- [ methyl-3H] glucose and [ 14C]mannitol. [3H]cytorat hindlimb muscles.To obtain sufficient muscle to prechalasin B was from Amersham. Sodium dantrolene was pare membrane fractions for the study of cytochalasin B obtained from Norwich Eaton Pharmaceuticals. Purified binding, we used the perfused rat hindquarter preparapork insulin was purchased from Squibb. Bovine serum tion. Perfusion of the hindlimb muscles for 40 min with albumin (Cohn Fraction V, fatty acid poor) was obtained hypoxic medium caused an increase in the rate of glucose from Miles Laboratories. All other reagents were pur- uptake to 20.5 t 1.1 pmol/min per hindquarter compared chased from Sigma. with 3.4 t 1.2 pmollmin per hindquarter for the controls (P < 0.01). (For comparison, the rate of glucose uptake in RESULTS rat hindquarters after 30-60 min of perfusion with oxy3-0-methylglucose transport. Hypoxia caused a large genated medium containing human erythrocytes at a concentration of hemoglobin of 12 g/100 ml in two stimulation of glucose transport activity in epitrochlearis muscles. As shown in Fig. 1, the increase in the rate of previous studies (15,45) averaged 3.3 t 0.3 pmol/min per 3-MG transport in response to hypoxia occurred over hindquarter for 33 rats.) The crude membrane fraction obtained from -30 g of -60 min, plateauing at a level approximately sixfold hindlimb muscle was subjected to discontinuous sucroseabove baseline. This hypoxia-induced increase in 3-MG uptake was due to activation of the glucose transport TABLE 1. Effects of cytochalasin B and of cycloheximide process and not to a nonspecific increase in permeability. on stimulation of 3-0-methylglucose transport by hypoxia This is evidenced by the findings that 1) 25 PM cytochalasin B completely inhibited 3-MG transport after 80 min in rat epitrochlearis muscle of hypoxia (Table 1) and 2) the muscles remained imper3-O-Methylglucose Transport, meable to mannitol. Cycloheximide (20 pg/ml), which inpm01 . ml ’ . 10 min.-’ Treatment I1 hibits protein synthesis in epitrochlearis muscle by 20 0.16+0.02 Control oxygenated -95% (46), had no effect on the increase in permeability 25 1.18t0.06 Hypoxia to 3-MG induced by hypoxia (Table 1). 0.0:3-to.o2 Hypoxia + 25 PM cytochalasin B 5 In the above experiments the muscles were allowed to Hypoxia -t 20 pg/ml cycloheximide 1.25kO.16 5 recover for 15 min in oxygenated medium, and 3-MG Values are means + SE and are expressed as pmol of 3-MG taken up transport was measured under aerobic conditions. When per ml of intracellular water in 10 min; IZ, no. of muscles. Epitrochlearis muscles were kept hypoxic throughout an 80-min-long muscles were incubated under oxygenated or hypoxic conditions with 8 incubation and the subsequent glucose washout and 3- mM glucose for 80 min at 35°C and were transferred to oxygenated MG transport measurement periods, the rate of 3-MG medium containing no glucose and incubated at 29°C for 15 min to wash out glucose and reoxygenate hypoxic muscles. Glucose transport transport was not significantly higher than in paired accumulation of muscles allowed to recover for 15 min in oxygenated me- activity was then assessed by measuring intracellular 3-0-methylglucose (3-MG) for 10 min at 29°C in oxygenated medium. dium before measurement of 3-MG transport (1.39 t In 1 experiment, cytochalasin B was present during wash period and 0.08 vs. 1.33 t 0.14 pmol . ml-’ 10 min-’ for 9 muscles measurement of 3-MG transport. In another experiment, cyclohexiincubations. per group). Thus the hypoxia-induced increase in glucose mide was present throughout
L 0
14.
l
l
l
l
l
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1596
STIMULATION
TABLE
OF
GLUCOSE
TRANSPORT
IN
MUSCLE
BY
HYPOXIA
2. Effects of hypoxia on epitrochlearis muscle ATP, phosphocreatine, and glycogen concentrations Treatment
pmol/g
Oxygenated control Hypoxia, 20 min Hypoxia, 80 min Reoxygenated
ATP, protein
Phosphocreatine, pmol/g protein
29.8t0.8 28.1k1.3 29.6k2.9
(18) (7) (5)
93.8+6.0 41.8t2.9* 32.2t3.7*
(18) (7) (5)
28.121.1
(11)
91.5k7.3
(11)
Glycogen, pmol/g protein
179+4 143+8 61-t12*
(8) (7) (5)
Values are means + SE for no. of muscles given in parentheses. Epitrochlearis muscles were incubated for 20 or 80 min under oxygenated or hypoxic conditions with 8 mM glucose at 35°C and then clamp-frozen. Additional muscles that had been incubated for 80 min under hypoxic conditions were transferred to oxygenated medium (reoxygenated) and incubated for 15 min before being frozen. Because there was no difference between values obtained on muscles incubated in oxygenated medium for 80 compared with 20 min, data obtained on these 2 groups were combined (oxygenated control). *Significantly less than oxygenated control (P < 0.01).
gradient centrifugation. The membrane fraction recovered floating on 25% sucrose is enriched approximately fivefold in the activities of the plasma membrane marker enzymes 5’-nucleotidase (Table 3 and Ref. ZO), Mg2+-ATPase (20)) and phosphodiesterase (20) relative to the crude membrane fraction, whereas the 35% sucrose fraction is relatively poor in these marker enzyme activities (Table 3 and Ref. 20). Sarcoplasmic reticulum Ca2+-ATPase activity was not detectable in either fraction. We have previously found that the number of glucose transporters, as measured by D-glucose-inhibitable cytochalasin B binding (20) and immunoblotting for the GLUT-4 glucose transporter (6), is increased in the 25% sucrose fraction and decreased in the 35% sucrose fraction from muscles treated with insulin. Taken together these findings provide evidence that the 25% sucrose fraction is enriched with plasma membrane, whereas the 35% sucrose fraction is poor in plasma membranes and probably contains an intracellular membranous organelle that, as in adipocytes (4,40), contains glucose transporters. Figure 2 shows the D-glucose-inhibitable cytochalasin B binding data expressed per milligram of protein; the relationships are the same when the data are expressed as the total amount of D-glucose-inhibitable cytochalasin B binding recovered in each fraction. Hypoxia resulted in an -50% increase in D-glucose-inhibitable cytochalasin B binding in the 25% sucrose (plasma membraneenriched) fraction, suggesting that hypoxia causes a translocation of glucose transporters into the plasma membrane in skeletal muscle. The difference in D-gh-
case-inhibitable cytochalasin B binding in the 35% sucrose fractions from the hypoxic and control muscles was not statistically significant. The effect of hypoxia on distribution of the GLUT-4 muscle glucose transporters in the plasma membrane and intracellular membrane fractions was also examined. Plasma membrane and intracellular membrane fractions prepared from control and hypoxic muscles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (30 fig protein from each fraction), transferred to nitrocellulose, and immunoblotted with anti-GLUT-4 antibody (Fig. 3). Hypoxia increased the amount of GLUT-4 transporters in the plasma membrane fraction but had little effect on the GLUT-4 content of the intracellular membrane fraction (Fig. 3). In two independent experiments, each analyzed twice, quantification by laser-scanning densitometry showed that hypoxia caused an average 2.4fold increase in GLUT-4 transporters in the 25% sucrose plasma membrane fraction without a decrease in GLUT-4 transporters in the 35% sucrose intracellular membrane fraction (Fig. 4). Interactions between hypoxia, muscle contractile activity, and insulin. As shown in Table 4, the maximal effects of in vitro muscle contractions or swimming on 3-MG transport were not significantly different from that of hypoxia. Furthermore, the combined effect of a maximal
20. 3. Protein content and 5’-nucleotidase activity in membrane fractions isolated from rat hindquarter muscles
TABLE
0 E a
5’-Nucleotidase, nmol min-’ protein-’ l
Protein,
mg/fraction
l
mg
Fraction
Control
Hypoxia
Control
Hypoxia
Crude membranes 25% Sucrose 35% Sucrose
42.2k6.2 1.26kO.13 ll.Ok1.8
38.5k5.2 1.2MO.08 10.3+_0.9
85+4 424k14 73+8
66+6 348+40 52+_5
Values are means k SE for 6 independent membrane preparations. Crude membrane fraction obtained from -30 g of rat hindlimb muscles was subjected to discontinuous sucrose gradient centrifugation (see METHODS). Aliquots of crude membrane preparation and membrane fractions recovered floating on 25% sucrose and on 35% sucrose were used for measurement of 5’-nucleotidase activity.
F ‘c1 1.0 c c1 m 0 00. 25% Membrane
3 5% Fraction
FIG. 2. Cytochalasin B (CB, 0.2 pM) binding to the 25% sucrose (plasma membrane) and 35% sucrose (internal membranes) fraction isolated from control and hypoxic muscles. Values are means + SE for 6 independent membrane preparations.
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STIMULATION
PM -II-II ’
OF GLUCOSE
IM
TRANSPORT
PM
IN MUSCLE
IM
1-- ,,’ :
43 kDa__>
r
._. \
“,
1597
BY HYPOXIA
\ __ _C_\\\_
CHCHCH
C
hypoxic stimulus and in vitro contractile activity on 3MG transport was not significantly different from that of either stimulus alone. Similarly, muscles taken from rats after a bout of swimming and then incubated under hypoxic conditions in vitro showed no greater increase in 3-MG transport than that induced by swimming alone (Table 4). This lack of additivity suggests that hypoxia and muscle contractions may activate glucose transport by the same mechanism. It has been shown previously (3, 29, 42) and again in this study (Table 5) that the maximal effects of exercise and insulin on glucose transport activity (i.e., increases above basal) are roughly additive in rat skeletal muscle. As would be expected if hypoxia and muscle contractions activate glucose transport by the same mechanism, the increases above basal in glucose transport activity induced by 60 min of hypoxia and a maximally effective insulin concentration were also additive (Table 5). Potentiation of a submaximal hypoxic stimulus by caffeine and nitrate. It has been hypothesized on the basis of
indirect evidence that the increase in cytoplasmic Ca2+ that occurs in response to stimulation of muscle contraction is the initiating event in the process that leads to activation of glucose transport by contractile activity
H
FIG. 3. Effect of hypoxia on subcellular distribution of the GLUT-4 transporter in skeletal muscle membranes. Plasma membranes (PM) and intracellular membranes (IM) were prepared from control (C) and hypoxic (H) rat hindlimb muscles. Membranes (30 pg protein) were separated on 12% polyacrylamide gels, transferred to nitrocellulose paper, and immunoassaved bv Western blot analvsis usine anti-GLUT-4 a&erum (see METHODS). Shown is an experiment with 2 independent membrane preparations.
(13). As a preliminary approach to evaluating the possibility that an increase in Ca2+ also mediates the activation of glucose transport by hypoxia, we examined the effects of caffeine and NO,. Caffeine induces release of Ca2+ from the sarcoplasmic reticulum (31) and, at sufficiently high concentration, causes muscle contraction and stimulation of glucose transport activity (13). In the rat epitrochlearis, a caffeine concentration above -3 mM is required to induce measurable tension development. Incubation of epitrochlearis muscles with 10 mM caffeine for 30 min induced an increase in 3-MG transport rate to 1.03 * 0.12 compared with a control value of 0.17 * 0.03 pm01 * ml-’ -10 min-’ (means + SE for 9 muscles per group); this is similar in magnitude to the response to a maximal hypoxic stimulus. Low concentrations of caffeine can elicit increases in cytosolic free-Ca2+ concentration that are below the contraction threshold (23). Incubation with 1 mM caffeine had no effect on 3-MG transport in oxygenated muscles but caused a significant potentiation of the effect of a submaximal (40-min-long) hypoxic stimulus on the rate of 3-MG transport without causing a contracture (Table 6) to give a sugar transport rate similar to that seen after a maximal hypoxic stimulus. However, 1 mM caffeine did not potentiate the effect of a maximal (80-min-long) hypoxic stimulus. NO, impairs the active uptake of Ca2+ by the sarcoplasmic reticulum and, as a consequence, potentiates muscle twitch tension (7). NO, also potentiates a submaximal effect of muscle contractions on glucose transport activity (14). Incubation of muscle in KHB in which 4. Effects of hypoxia and muscle contractile activity together on 3-O-methylglucose transport TABLE
Treatment
PM
IM membrane
fraction
FIG. 4. Effect of hypoxia on subcellular distribution of GLUT-4 transporters in skeletal muscle PM and IM preparations. OD, optical density. Values are expressed as a relative increase in response to hypoxia. Two independent membrane preparations were analyzed. Each preparation was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoassay by Western blot analysis twice. Each blot was developed twice, and each X-ray film was densitometritally scanned twice. Values are averages f SD of these determinations.
Oxygenated control Hypoxia In vitro muscle contractions Hypoxia + muscle contractions Swimming Swimming + hypoxia
It
11 11 12 9 17 15
3-0-Methylglucose jmol~ ml-‘.
Transport, 10 min-’
0.21*0.02 1.18+0.10 1.15rto.07 1.34kO.08 1.40+0.14 1.31+0.13
Values are means + SE; n, no. of muscles. Epitrochlearis muscles were incubated, under oxygenated or hypoxic conditions, with 8 mM glucose for 60 min at 35°C. In 1 series of experiments, muscles were transferred to oxygenated medium for 5 min and then stimulated to contract (see METHODS). In a second series of experiments, muscles were from rats that had been exercised by means of swimming immediately before study.
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1598
STIMULATION
OF
GLUCOSE
TRANSPORT
Oxygenated control Hypoxia 2,000 PUlml insulin Hypoxia + 2,000 pU/ml insulin Swimming Swimming + 2,000 &J/ml insulin
n
12 12 8 8 6 6
O.ZOrtO.03 1.2O-tO.08 1.26kO.09 2.22+0.13* 1.22+0.15 1.98*0.09j-
NaNO, was substituted for NaCl had no effect on glucose transport activity when the medium was oxygenated (Table 6). However, NO, significantly potentiated the effect of a submaximal (40 min) hypoxic stimulus on glucose transport (Table 6). The maximal response to hypoxia was unaffected by NO,. Effect of dantrolene. Dantrolene inhibits release of Ca2+ from the sarcoplasmic reticulum in skeletal muscle (7). As shown in Table 7, 25 PM dantrolene partially inhibited the hypoxia-induced stimulation of glucose transport activity. The increase in 3-MG transport rate in response to 60 min of hypoxia was
